Breastfeeding does not raise the risk of mother-to-child transmission of hepatitis C virus (HCV), according to two new studies published in the December 1 issue of The Journal of Infectious Diseases, now available online.

One study found that infant girls are twice as likely to be infected as infant boys. Both studies provide new information with which to counsel pregnant women infected with HCV. Taken together, the two new studies expand upon preliminary data from smaller studies of mother-to-child transmission of HCV.

The larger of the two studies, conducted by the European Paediatric Hepatitis C Virus Network, involved 1,479 mother-and-child pairs enrolled at 33 centers in Italy, Spain, Germany, Ireland, the United Kingdom, Norway, and Sweden. The other study, by Eric E. Mast, MD, MPH, of the U.S. Centers for Disease Control and Prevention and colleagues, followed 244 infants born to infected mothers in Houston and Honolulu.

The finding of gender differences in HCV infection was reported by the European authors, who hypothesized that their result may reflect hormonal or genetic differences between men and women in susceptibility or response to infection. Other risk factors significantly associated with transmission were the time in labor (a risk factor in both studies) and use of internal fetal monitoring devices (a risk factor in the U.S. study only).

Although breastfeeding is a known risk for HIV transmission, both studies found it was not associated with transmission of HCV. The European study also found that caesarean section delivery, infant prematurity, and maternal history of injection drug use were not associated with HCV transmission.

The overall rate of transmission of the virus from infected mother to child was 6.2 percent in the European study and 3.6 percent in the U.S. study.

"Our results strongly suggest that women should not be offered an elective caesarean section or discouraged from breastfeeding on the basis of hepatitis C infection alone," said Pier-Angelo Tovo, MD, the lead author of the European study.

"Our findings support existing recommendations to avoid internal fetal monitoring and prolonged laborâ¦in infected women," wrote the U.S. authors.

In an accompanying editorial, R. Palmer Beasley, MD, of the University of Texas at Houston, emphasized the European study's novel finding of a gender difference in transmission rates and suggested that higher HCV rates in female newborns may be due to excess mortality in infected males in utero. "Overall, the observation of higher hepatitis C virus infection rates in female infants is in accord with recent observations of similar excesses in HIV infection of female infants," Dr. Beasley said.

Founded in 1904, The Journal of Infectious Diseases is the premier publication in the Western Hemisphere for original research on the pathogenesis, diagnosis, and treatment of infectious diseases; on the microbes that cause them; and on disorders of host immune mechanisms. Articles in JID include research results from microbiology, immunology, epidemiology, and related disciplines. JID is published under the auspices of the Infectious Diseases Society of America (IDSA). Based in Alexandria, Va., IDSA is a professional society representing about 8,000 physicians and scientists who specialize in infectious diseases. For more information, visit www.idsociety.org.

EDITORIAL
Nature Usually Favors Females

R. Palmer Beasley
School of Public Health, University of Texas, Houston

This issue of the Journal treats us to articles from 2 large multicenter studies of vertical transmission of hepatitis C virus (HCV). The studies are complementary and have largely similar findings, although the European study [1], with 1787 mother-infant pairs of HCV-positive mothers from 33 centers in 7 countries, dwarfs the American one [2], which included 244 mother-infant pairs from 2 centers. Overall, the estimated frequencies of HCV transmission were 6.2% (95% confidence interval [CI], 5.0%7.5%) in the European study and 3.6% (95% CI, 1.3%6.0%) in the American study. Both studies found an increased risk of transmission from mothers with higher viral loads, earlier rupture of membranes, and coinfection with HIV. Neither study found a correlation between transmission and either breast-feeding or cesarean-section delivery. Also of interest is that both studies found that a small proportion of apparently infected infants subsequently lost all HCV markers on longer follow-up.

The most interesting observation from the European study is that HCV infection was twice as frequent among female than male infants (unadjusted odds ratio, 2.03 [95% CI, 1.303.15]; P = .001). The difference was similar in the American study (6 girls and 3 boys) but was not statistically significant, because of the much smaller sample sizes. The European investigators indicate that these are probably the first data suggesting a differential response to HCV between males and females. Sex differences are found in responses to most infectious agents, but these findings are interesting because there are more HCV infections among the female than the male infants. This is quite remarkable, since a male excess is found for most infectious agents, and, in general, males respond more poorly than females to most external challenges. Males have a shorter life expectancy, and males of all age groups have higher overall mortality than females. Except for female end-organ disease and those
illnesses explained by exposure differentials (e.g., skin cancer), there are relatively few conditions in which the incidence in females exceeds that in males. There are autoimmune diseases and a few cancers (e.g., of the thyroid and gall bladder) that are more common in females, but far and away most diseases, including infectious diseases, have a male excess. The extra X chromosome appears to be protective.

It is perplexing that the European investigators provide little information to help interpret the sex differences they observe and emphasize in the title of their article. They suggest that it "reflects hormonal or genetic differences in susceptibility or response to infection between males and females" (p. XXX), but they have not provided enough information to give reasonable assurance that these differences are not due to bias or confounding. However, if bias and confounding can be eliminated, then I believe that the most likely possibility is excess male mortality in utero rather than increased infections among females. The authors can help us interpret their findings by providing the complete sex distribution at birth of the infants in their study. Also absent is information about abortion rates, infection, and follow-up rates by sex at different ages, which must be available, since their protocol called for testing of infants at birth, age 6 weeks, and ages 3, 6, 9, 12, 18, and
24 months, as well as some beyond that. These deficiencies make it unnecessarily difficult to analyze the various possible interpretations.

If the sex ratio at birth in this study were decreased from the 1.05 (male : female) ratio found in the general populations of most Western countries, it would strongly suggest an adverse in utero effect on males. A decreased ratio was observed in the American study in Houston (the Hawaii component data were not available), where the birth sex ratio was 99 : 113, or 0.876. The sex ratio at enrollment in the European study was 668 : 802, or 0.833, which is probably the birth ratio, but the authors, unfortunately, do not define enrollment, so we cannot be certain what biases were introduced if the data were censored. In view of these numbers showing a low male : female ratio, I am unable to interpret the following statement by the European authors, which seems at odds with these data of theirs: "Because the male : female ratio in our study population was higher than that observed in the general population, this finding is unlikely to be due to excessive deaths of infected males in
utero" (p. XXX).

For hepatitis B virus (HBV), males of all ages have slightly higher infection rates than females and slightly poorer responses (titers and conversion rates) to HBV vaccination, and there appears to be no distortion of the sex ratio at birth of infants born to carrier mothers. The incidence of chronic liver manifestations of the HBV carrier state (chronic hepatitis, cirrhosis, and hepatocellular carcinoma) are much more common in males than females, at ratios of 2.53.5, depending on the population. Intrauterine infections make up a small proportion of perinatal HBV infections, so it is conceivable that a differential adverse sex effect in that small proportion could have been missed by such relatively crude measures as sex ratios at birth.

The authors of the European study reference 3 HIV perinatal transmission studies, which found higher frequencies of HIV infection in female infants and lower male : female birth ratios. Taha et al. [3] reported data from a large vertical transmission study in Malawi, which showed HIV positivity of 12.6% among 998 girls, compared with 6.3% among 966 boys, a male : female ratio at birth of 0.968 for enrolled infants. A final observation from the Malawi study deserves comment. Female infants had a smaller average birth weight than male infants, as would be expected if there were more in utero fetal deaths among males, if the infections were more common in the smaller and/or weaker ones.

In conclusion, the observation of higher HCV infection rates in female infants of HCV-infected mothers is interesting, provocative, and worth further investigation. It is in accord with recent observations of similar excesses of HIV infections in female infants of HIV-infected mothers. Future studies investigating infant sex differences in relation to perinatal infections need to plan carefully to collect and present sex data in a fashion that minimizes potential bias, such as skewed abortions and stillbirths, if sex is known before birth, and distorted follow-up by sex.

Risk Factors for Perinatal Transmission of Hepatitis C Virus (HCV) and the Natural History of HCV Infection Acquired in Infancy

1Centers for Disease Control and Prevention and 2Emory University, Atlanta, Georgia; 3University of Texas Houston School of Public Health, Houston; 4Kapiolani Medical Center and University of Hawaii School of Medicine, Honolulu

ABSTRACT
Background. The goal of the present study was to assess risk factors for perinatal hepatitis C virus (HCV) transmission and the natural history of infection among HCV-infected infants.

Methods. In a cohort study, 244 infants born to HCV-positive mothers were followed from birth until age >12 months. Maternal serum was collected at enrollment and delivery; infant serum was collected at birth and at 8 well-child visits. Testing included detection of antibody to HCV, detection of HCV RNA (qualitative and quantitative), and genotyping. HCV-infected infants were followed annually until age 5 years.

Results.

Overall, 9 of 190 (4.7% [95% confidence interval {CI}, 2.3%9.1%]) infants born to mothers who were HCV RNA positive at delivery became infected, compared with 0 of 54 infants born to HCV RNAnegative mothers (P = .10). Among HCV RNA positive mothers, the rate of transmission was 3.8% (95% CI, 1.7%8.1%) from the 182 who were human immunodeficiency virus (HIV) negative, compared with 25.0% (95% CI, 4.5%64.4%) from the 8 who were HIV positive (P < .05).

Conclusion. If duration of membrane rupture and internal fetal monitoring are confirmed to be associated with transmission, interventions may be possible to decrease the risk of transmission.

DISCUSSION
The rates of HCV transmission to infants from HCV-infected, HIV-negative mothers (3.8%) and from HCV/HIV-coinfected mothers (25%) in the present study are consistent with findings from other studies [2]. Our results provide information that can be used for counseling regarding the risk of transmission through breast-feeding, the timing of follow-up to distinguish infected from uninfected infants, and the course of infection during the first 5 years of life. The study also provides insight into risk factors that might facilitate and interventions that might prevent perinatal HCV transmission.

Transmission occurred only from mothers who were HCV RNA positive, which is consistent with findings from other studies. The only 2 documented episodes of transmission from HCV RNAnegative mothers [11, 12] could be explained as resulting from the use of testing methods that were not sensitive enough to detect low levels of HCV RNA or from intermittent HCV RNA detection in an HCV-infected pregnant woman [13]. Although we detected HCV RNA intermittently among some women, >96% had consistent results when tested both prenatally and at delivery, indicating that a single HCV RNA test during pregnancy should be sufficient for diagnosis and counseling. Maternal HCV RNA testing should be required in studies of perinatal HCV transmission, and analyses should be restricted to HCV RNApositive mothers or stratified by HCV RNA status.

Although higher maternal viral levels increase the risk of perinatal transmission of HIV and hepatitis B virus (HBV) [1416], data on associations between maternal HCV RNA level and perinatal transmission have been inconsistent. In univariate analysis of HCV-positive, HIV-negative mothers, the risk of transmission has been found to increase with increasing maternal HCV RNA level, and mean levels were found to be higher in transmitting than in nontransmitting mothers. However, maternal HCV RNA level was not independently associated with transmission in multivariate analysis among HIV-negative women. A direct relationship between maternal viral level and increased transmission rates has been reported by some investigators [4, 5, 8, 1720] but not by others [10, 21, 22]. Furthermore, among the studies reporting an association, the threshold viral level associated with transmission has differed. Reasons for the inconsistencies could include the use of different methods to quantify HCV RNA
levels, combining HIV-negative and HCV/HIV-coinfected women in analyses, and unrecognized statistical interactions between viral level and other factors. Given the inconsistency of results and the evidence for transmission from women with HCV RNA levels <106 genome copies/mL, we believe that HCV RNA level cannot be used to counsel HCV RNApositive women about their risk for perinatal HCV transmission.

Perinatal HCV transmission could occur in utero, during the intrapartum period, or postnatally. We found no evidence for postnatal transmission, because all infected infants were HCV RNA positive by age 2 months, which is consistent with HCV exposure occurring at or before the time of delivery. This finding contrasts with the pattern of mother-to-child HBV transmission, in which as many as 40% of infants born to HBV-infected mothers who are not infected during the intrapartum period become infected during the first 18 months of life [23]. In addition, breast-feeding was not associated with transmission, which is consistent with findings of other studies [3, 12, 19, 22, 2427] and provides further support for recommendations that HCV-positive, HIV-negative women can safely breast-feed [28, 29]. Differentiation of in utero from intrapartum transmission is primarily based on detection of HCV RNA at the time of birth. Although HCV RNA was detected in peripheral venous serum samples from
some infected infants at birth by use of nested PCR in the present study, HCV RNA was also detected in uninfected infants, suggesting that HCV RNA positivity at birth may reflect passive transferral of maternal virus rather than intrauterine transmission.

Recommendations for screening and follow-up of infants born to HCV-infected mothers include anti-HCV testing at age >15 months or nucleic acid testing on 2 occasions between ages 2 and 6 months [30]. Our findings suggest that it may be prudent to delay anti-HCV testing until age >18 months, because 2 infected infants who were anti-HCV negative at 15 months subsequently seroconverted to anti-HCV positivity, one between age 15 and 18 months and the other between age 18 and 24 months. Testing at age >18 months also eliminated all false-positive results in infants with passively acquired anti-HCV, if RIBA testing was used to verify anti-HCV EIA screening testpositive results [31]. Our findings support the recommendation for nucleic acid testing, because all infected infants were HCV RNA positive at ages 2, 4, and 6 months. Testing before age 2 months cannot be recommended, because detection of HCV RNA in both cord and peripheral venous serum samples collected at birth is likely to
indicate contamination with maternal blood or passive transfer of maternal HCV RNA, rather than infant infection.

Although none of the infected infants in this study had clinical evidence of hepatitis during 5 years of follow-up, all had abnormal ALT levels at some point. These findings indicate that liver disease among HCV-infected infants is generally mild [32, 33]. The higher proportion of HCV-infected infants in the present study who resolved their infections, compared with those in follow-up studies of persons infected at older ages [5, 3235], demonstrates the importance of follow-up testing of HCV-infected infants to determine the clinical progression of infection.

Among infants born to HCV-infected, HIV-negative mothers, longer duration of membrane rupture and invasive fetal monitoring were associated with transmission. Of 2 studies that evaluated duration of membrane rupture [5, 36], 1 found increased transmission rates associated with longer duration [5]. One study found increased transmission rates associated with fetal scalp monitoring [37], and another found increased transmission rates associated with intrapartum exposure to maternal blood [4]. One hypothesis to explain these findings is that perinatal transmission generally occurs during the intrapartum period and is related to infant exposure to maternal genital tract secretions or blood. Although one study found no evidence of virus in genital tract secretions [38], another detected virus in the cellular fraction of cervicovaginal secretions of 27% of infected women [39]. Both longer duration of exposure of infant mucous membranes and percutaneous inoculation of the infant could
enhance transmission.

Our findings suggest that avoiding internal fetal monitoring and/or perfoming cesarean section before or soon after membrane rupture could decrease the risk of perinatal transmission from HCV-infected mothers who were identified prenatally. However, results from studies comparing risk for HCV transmission among infants delivered vaginally and infants delivered by cesarean section are conflicting [2, 3, 11, 12, 18, 26]. In addition, in virtually all of these studies, most study participants were HCV/HIV coinfected, coinfected and HIV-negative women were combined for analysis, and, in some instances, other potential risk factors were not accounted for. These features make it difficult to determine whether the results apply to infants born to women infected only with HCV. In addition, only 1 study differentiated elective cesarean sections performed before membrane rupture from emergency cesarean sections; that study found a lower transmission rate from mothers who delivered by elective
cesarean section than from mothers who delivered by emergency cesarean section, although the analysis also included HCV/HIV-coinfected women [3]. Among HIV-infected women, elective cesarean section performed before onset of labor and rupture of membranes not only was associated with lower HIV transmission rates but also was shown, in a randomized trial, to be an effective intervention [40, 41]. In our study, no transmission occurred from mothers who delivered by elective cesarean section, but the number of such deliveries was low.

Our findings support existing recommendations to avoid internal fetal monitoring and prolonged labor after rupture of membranes in HCV-infected pregnant women [30]. Current recommendations regarding the need for cesarean section versus vaginal delivery are not based on HCV infection status. Any changes in cesarean section practices for HCV-infected pregnant women should be considered cautiously and should be based on separate studies of HCV/HIV-coinfected women and women infected only with HCV. Ideally, prospective studies should be conducted to determine whether elective cesarean section delivery reduces the risk for perinatal HCV transmission.

Methods. We conducted a European multicenter prospective study of HCV-infected pregnant women and their infants. Children with 2 positive HCV RNA polymerase chain reaction test results and/or anti-HCV antibodies after 18 months of age were considered to be infected.

Conclusions. Our results strongly suggest that women should neither be offered an elective CS nor be discouraged from breast-feeding on the basis of HCV infection alone. The sex association is an intriguing finding that probably reflects biological differences in susceptibility or response to infection.

DISCUSSION
HCV infection is a major public health problem and is of particular concern during pregnancy. Identification of transmission risk factors is essential for the development of appropriate interventions. In this large, prospective European cohort, girls were twice as likely to be infected as boys. There was no evidence of a protective effect of elective CS delivery. However, the duration of ROM was significantly longer among mothers of infected children than among mothers of uninfected children. HCV/HIV-coinfected women were more likely to transmit HCV to their infants than were women with HCV infection only, although this was not statistically significant in multivariable analysis. An increased but not statistically significant risk of transmission was also observed among women who were HCV viremic during pregnancy or around the time of delivery, although a few children were infected by nonviremic women. Maternal history of IDU, prematurity, and type of infant feeding were not
significantly associated with transmission.

To our knowledge, this is the first report of a significant association between sex and HCV vertical transmission. Granovsky et al. reported an 8% transmission risk for girls, compared with 3% for boys, in a study of 122 mother-child pairs with 7 infected children, but this difference was not statistically significant [14]. A significantly higher susceptibility of females to vertically acquired infections has been observed for human T cell leukemia/lymphoma virus type I [15] and for HIV in Europe [16] and in Malawi [17], although the risk of postnatal HIV transmission was found to be significantly higher in males in a meta-analysis of African trials [18]. Because the male : female ratio in our study population was higher than that observed in the general population, this finding is unlikely to be due to excessive deaths of infected males in utero. It likely reflects hormonal or genetic differences in susceptibility or response to infection between males and females. Maternal
infections increase fetal cortisol and dehydroepiandrosterone synthesis [19]. Androgens may influence the immune response [20]; estradiol protects stimulated feline lymphocytes from apoptosis [21], and human male and female fetuses exhibit differences in regulation of the cytokine network [22]. Sex differences in mortality associated with routine childhood modified virus vaccinations may also indicate differential susceptibility to infection [23].

Until recently, maternal HCV/HIV coinfection was consistently found to be associated with an increased risk of HCV vertical transmission [4, 6, 7], thought to be mediated by the higher HCV load in immunosuppressed HCV/HIV-coinfected women than in women with HCV infection only. The attenuation of this association seen in the present study is likely to be due to the recent widespread use of HAART, which substantially improves HIV-induced immunosuppression and, consequently, may lead to better control of HCV replication; a similar finding was reported in a small study involving 15 HCV/HIV-coinfected ART-treated women [24]. Our results suggest that HAART may reduce both maternal HCV load and risk of HCV transmission. Since HAART causes prematurity [13], it can also account for the apparent protective effect of prematurity on HCV vertical transmission, seen only in HCV/HIV-coinfected women.

Although the risk of vertical transmission is likely to be substantially lower in nonviremic women, our results confirm our previous finding [9] that it cannot be ruled out, since a few children acquired infection from nonviremic women. Data on maternal HCV viremia must be interpreted with caution, since fluctuations may occur during pregnancy [25]. The results of 1 or 2 PCR tests during pregnancy or at delivery do not necessarily reflect viremia status at the relevant time for transmission. Viral load may be a better predictor of transmission than a qualitative test. However, even where viral load data are available, interpretation is problematic, especially in multicenter studies, because of interlaboratory and interassay variations [26].

The proportion of elective CS was high in our study among HCV/HIV-coinfected women, for whom a targeted recommendation exists [10], as well as among those with HCV infection only, especially in Italian centers, where this practice is common among the general population [11]. Our current findings make an important contribution to the debate [4, 5, 24, 2729] and strongly suggest that mode of delivery does not influence HCV transmission in women with HCV infection only or in HCV/HIV-coinfected women when they are receiving potent ART. This study would have been large enough to detect a 60% reduction in risk of transmission associated with elective CS delivery, from 6% to 2.5% (with 80% power and 5% significance). Consistent with the findings of Spencer et al. [30], it is of note that, despite the lack of a protective effect of elective CS, the duration of ROM was related to transmission risk. Finally, our results confirm [6, 12, 30, 31] that breast-feeding does not increase the risk of
HCV transmission, and, unlike Resti et al. [12], we did not observe an association between history of maternal IDU and HCV vertical transmission.

In observational studies, there is always a concern that unobserved factors may exist that could be related to the outcome of interest. However, although the goodness of fit of our models improved after the inclusion of a random effect in multivariable modelssuggesting that there may be small center-associated differences in vertical transmission riskthe estimated associations between the variables of interest here and the risk of HCV vertical transmission did not change.

What are the implications of our findings for the clinical management of HCV-infected women? The overall risk of vertical transmission is low for women with HCV infection only but cannot be ruled out even in nonviremic mothers. Given that there is no antiviral treatment available for use during pregnancy, there are currently no interventions to prevent mother-to-child HCV transmission. In particular, there is no evidence to suggest that women should be offered an elective CS delivery or be advised to avoid breast-feeding. HCV/HIV-coinfected women are currently treated with HAART during pregnancy to prevent HIV vertical transmission; this treatment may also have a beneficial effect on HCV vertical transmission.